The overall goal of this research proposal is to further define the role of the Abcg2 transporter in hematopoietic stem cells (HSCs), in stem cells from other non-hematopoietic organs, and in cancer stem cells. Work in the prior funding period has shown that: 1) Abcg2 expression is required for the side population (SP) phenotype in HSCs and serves as a prospective molecular marker for isolating HSCs, 2) Abcg2-expressing SP cells exist in skeletal muscle, and as demonstrated by other labs, in many other non-hematopoietic organs, 3) a majority of human AML samples contain a small subpopulation of ABCG2-expressing cells that could represent a leukemia stem cell population, and 4) expression of Abcg2 protects HSCs from mitoxantrone, a xenobiotic drug. Experiments are now planned that will unequivocally determine whether Abcg2-expressing cells have stem cell activity in skeletal muscle and in other organ systems. By using a novel genetic approach, we will track the progeny of Abcg2-expressing cells in mice at different stages of development in order to evaluate candidate organ systems for the presence of Abcg2-expressing stem cells. A second aim is to use monoclonal antibodies to isolate hematopoietic stem cells from human and non-human primates. Using a panel of monoclonal antibodies that recognize external epitopes on ABCG2 that are present on living cells, we will isolate cells expressing ABCG2 and test them for hematopoietic stem cell function, with the ultimate goal defining a new stem cell population for use in transplantation protocols. Lastly, we will evaluate whether Abcg2 expression can identify cancer stem cells in myelogenous leukemia and in neuroblastoma. Both tumors contain a subpopulation of cells that express the side population phenotype and ABCG2, and therefore may represent a cancer stem cell population. We have and will develop novel genetic mouse models that should allow us to test this hypothesis in an unequivocal fashion. Not only could this provide a new marker for cancer stem cells, but it would have prognostic and therapeutic significance for these human cancers. Altogether, these studies should further elucidate the role of the Abcg2 transporter in normal and malignant stem cells, and should further increase our understanding of the role of ABC transporters in stem cells.